Beta-adrenergic agonists increase the myocardial contraction amplitude and enhance relaxation. 0ur studies examine the autonomic modulation of the calcium-myofila-ment interaction to determine whether it could account for enhanced twitch relaxation. In single cardiac myocytes we found that for a given amplitude of-cytosolic calcium twitch amplitude was less in isoproterenol (Iso) than in control. In myocytes suspensions norepinephrine (NE, 10 uM) increased troponin-I phosphorylation fourfold and under similar conditions decreased twitch relaxation time in individual myocytes by 20%. After propranolol (1.0 uM) troponin slowly dephosphorylated (half time 16.9+1.7 min) but relaxation time fully returned to control within 5 min. Thus, the -extent of troponin-I phosphorylation by NE is not directly proportional to its effect on relaxation time. In isolated, aequorin-injected ferret cardiac muscle the calcium tension relation was determined from the peak aequorin luminescence and peak tension measured across a broad range of bathing calcium in the presence and absence of acetyl-choline (ACh, I uM) or Iso (1 uM) or both drugs. Ach shifted the relationship of peak tension to (peak) aequorin light leftward, suggesting an increase in myofila-ment calcium sensitivity, but did not alter twitch relaxation (tl/2R). Iso shifted the relationship of peak tension to peak aequorin light rightward and decreased tl/2R. Ach added to Iso abolished the Iso effect on peak tension-aequorin light relationship but did not reverse the effect of Iso to decrease tl/2R. Thus per-turbations of the apparent myofilament calcium interaction in the intact muscle do not necessarily relate to twitch relaxation time.